CN110974743A - Method for producing yeast cycloastragenol polypeptide mixed liquor and anti-aging application thereof - Google Patents
Method for producing yeast cycloastragenol polypeptide mixed liquor and anti-aging application thereof Download PDFInfo
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Abstract
The invention discloses a method for producing yeast cycloastragenol polypeptide mixed liquor and anti-aging application thereof, wherein the method comprises the steps of mixing an astragalus extract with crude enzyme liquid of a Kluyveromyces lactis mutant strain XT 82; stirring for reaction to obtain reaction liquid; then adding water into the reaction liquid for dilution, then cooling to 20-60 ℃, and then sequentially adding carnosine, palmitic acid pentapeptide and acetyl hexapeptide-8; obtaining mixed solution of yeast cycloastragenol polypeptide; the application of the yeast cycloastragenol polypeptide mixed liquor in preparing anti-aging products.
Description
Technical Field
The invention relates to the field of biotechnology and cosmetics, in particular to a method for producing yeast cycloastragenol polypeptide mixed liquor and anti-aging application thereof.
Background
As a traditional Chinese medicine, Huang Qi is widely used up to now because it has the actions of tonifying Qi and spleen, raising Yang and sinking, benefiting Wei and consolidating exterior. Cycloastragenol is a cycloartane tetracyclic triterpenoid compound, which is mainly present in astragalus in the form of aglycone in nature and is reported to be prepared from astragalus index component-astragaloside, and the research of U.S. GeRON biotechnology company in 2010 discovers that cycloastragenol mainly plays a biological role by activating telomerase, and the discovery actively promotes the research work of cycloastragenol, and then researchers discover that the cycloastragenol has various pharmacological activities such as delaying senescence, enhancing T cell capacity and inhibiting pulmonary fibrosis, which are related to the effect of activating telomerase.
Cycloastragenol hardly exists in nature, and is obtained by hydrolyzing astragaloside IV in radix astragali in intestinal tract by microorganism to remove xylose at C3 and glucose at C6. Therefore, the hydrolysis of astragaloside is necessary to obtain cycloastragenol, and the hydrolysis method is a core technology. At present, the chemical method can completely hydrolyze astragaloside by using strong acid to obtain cycloastragenol, but the environmental cost is high, and the final product can be used in the field of food or cosmetics only after being subjected to a plurality of steps such as neutralization, purification and the like. The biological method utilizes microorganism (producing characteristic glycosidase) or glycosidase to hydrolyze astragaloside, can also obtain cycloastragenol, and has the advantages of mild process, less byproducts and the like. However, microorganisms used in the reported patents are often genetic engineering bacteria such as escherichia coli and the like, which not only do not meet the safety requirements of foods and cosmetics, but also relate to safety problems such as transgenosis and the like; the reported enzymatic hydrolysis method is generally carried out at normal temperature, has low reaction efficiency and long time period, and is easy to pollute microorganisms in the production process.
Therefore, a method for rapidly degrading cycloastragenol obtained by astragaloside with food safety at high temperature is urgently needed in the market.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for producing yeast cycloastragenol polypeptide mixed liquor and anti-aging application thereof. The method comprises the steps of preparing a cell lysis extract by using Kluyveromyces lactis XT82 strain, carrying out hydrolysis reaction on the cell lysis extract and an astragalus extract at high temperature, and adding three polypeptides of carnosine, pentapeptide palmitate and acetyl hexapeptide-8 to obtain a yeast cycloastragenol polypeptide mixed solution.
In order to achieve the aim, the invention designs a method for producing a yeast cycloastragenol polypeptide mixed solution, which comprises the following steps:
1) inoculating Kluyveromyces lactis mutant strain XT82 into YPD liquid culture medium for culture and activation; then transferring to YPD liquid culture medium for fermentation (culture conditions are that temperature is 30 deg.C, rotation speed is 250rpm, culture time is 24-96 hr, wherein 36-84 hr is preferable, 48-72 hr is most preferable), centrifuging and collecting upper cell; wherein the Kluyveromyces lactis mutant strain XT82 is named as Kluyveromyces lactis XT82, and is preserved in the China center for type culture Collection in 2019, 16 months, 9 months, the preservation number of the Kluyveromyces lactis university in Wuchang Lojia mountain of Wuhan city, Hubei province is CCTCCNO: m2019724;
2) washing the collected cells with deionized water for 2-3 times, dissolving in deionized water to prepare suspension cells, crushing the cells, centrifuging, collecting supernatant to obtain crude enzyme solution
3) Weighing the astragalus extract and the crude enzyme solution according to the weight-volume ratio of 40-60 g/L; stirring for reaction to obtain reaction liquid;
4) then adding water into the reaction liquid for dilution, then cooling to 20-60 ℃, and then sequentially adding carnosine, palmitic acid pentapeptide and acetyl hexapeptide-8; obtaining mixed solution of yeast cycloastragenol polypeptide; wherein, in the mixed solution of yeast cycloastragenol polypeptide, the content of cycloastragenol is 0.01-0.02%,
the content of carnosine is 50-200ppm,
the content of the palmitic acid pentapeptide is 50-200ppm,
the content of acetyl hexapeptide-8 is 50-200 ppm.
Further, in the step 1), the YPD liquid medium is prepared by the following steps:
sterilizing 10 g of the fermented extract and 20 g of peptone in 900ml of deionized water at 121 ℃ for 20 min; after cooling to about 60 ℃, 50ml of filtered and sterilized 40% glucose solution is added and mixed evenly.
Furthermore, in the step 3), the weight volume ratio of the astragalus extract to the crude enzyme solution is 40-60 g/L; wherein the content of astragaloside IV in the radix astragali extract is 4-6%.
Still further, in the step 4), the stirring reaction conditions are as follows: the temperature is 70-80 ℃ and the time is 1-3 h.
Still further, in the step 4), the volume ratio of the reaction liquid to water is 1: 5-100.
Still further, in the step 4), the volume ratio of the reaction liquid to water is 1: 10-50 parts of; the temperature of the reaction solution was reduced to 20-45 ℃.
Still further, in the step 4), the volume ratio of the reaction liquid to water is 1: 15-30 parts of; the temperature of the reaction solution was reduced to 25-40 ℃.
Furthermore, in the mixed solution of the yeast cycloastragenol and the polypeptide, the content of the cycloastragenol is 0.01 percent; the content of carnosine is 100 ppm; the content of the palmitic acid pentapeptide is 50 ppm;
the content of acetyl hexapeptide-8 was 50 ppm.
The invention also provides an application of the yeast cycloastragenol polypeptide mixed liquor in preparing an anti-aging product.
The invention has the beneficial effects that:
1) the invention utilizes the Kluyveromyces lactis mutant strain XT82 to produce yeast cycloastragenol polypeptide mixed solution; the Kluyveromyces lactis XT82 can secrete a broad-substrate-spectrum glycosidase, and can efficiently hydrolyze glucoside, galactoside, xyloside, arabinoside and the like at 75 ℃. And is excellent in thermal stability. Experiments show that the glycosidase can efficiently and rapidly hydrolyze all the cycloastragenol obtained from astragaloside IV at high temperature (70-80 ℃) (within 2 hours), has no byproducts, does not need organic solvents, is environment-friendly, and does not pollute other microorganisms at high temperature. The cycloastragenol product obtained by hydrolysis by the method can be directly used in the fields of food and cosmetics.
2) The invention utilizes Kluyveromyces lactis mutant strain XT82 to efficiently and specifically hydrolyze astragaloside IV under the condition of high temperature to obtain the cycloastragenol, the mother solution of the strain is rich in various amino acids, polypeptides, vitamins, trace elements and the like besides the high-concentration cycloastragenol, the mother solution is a brand-new, high-nutrition and safe cosmetic raw material, and the product has the effects of activating the telomerase activity of epidermal cells, delaying skin aging, fading static lines and dynamic lines of the epidermis, resisting oxidation, supplementing nutrient elements required by skin metabolism and the like, and has extremely high commercial value.
Drawings
FIG. 1 is a graph of data on the temperature stability of a yeast cycloastragenol polypeptide mixture;
FIG. 2 is an electron microscope image of yeast cycloastragenol polypeptide mixture used for improving skin texture imbalance experiment;
FIG. 3 is a diagram showing the effect of yeast cycloastragenol polypeptide mixture for lightening fine lines of facial epidermis;
FIG. 4 is a graph showing the effect of yeast cycloastragenol polypeptide mixture on improving epidermal cytochrome and elasticity.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1 fermentation Process of Kluyveromyces lactis XT82
Inoculating the preserved Kluyveromyces lactis XT82 into 3-10ml YPD liquid culture medium, and continuously culturing at 30 deg.C and 250rpm for 48 hr; transferring the strain into 1-10L YPD liquid culture medium according to the inoculation amount of 1-5%, and continuously culturing for 48-72 hours at the temperature of 30 ℃ and the rotating speed of 250 rpm; after the culture is finished, centrifuging for 10-30min at the rotating speed of 3000-; wherein, YPD liquid culture medium:
sterilizing 10 g yeast extract and 20 g peptone in 900ml deionized water at 121 deg.C for 20 min; after cooling to about 60 ℃, 50ml of filtered and sterilized 40% glucose solution is added and mixed evenly.
EXAMPLE 2 preparation of crude enzyme solution
The cells collected in example 1 were washed 2-3 times with deionized water. Adding 0.05-0.5L deionized water, suspending the cells, and crushing the cells under the pressure of 1500-2000bar of a high-pressure homogenizer. And (3) preserving the temperature of the crushed cells at 95 ℃ for 30 minutes, centrifuging the cells for 30 minutes at 10000rpm after complete denaturation of the protein with intolerance to high temperature, collecting supernatant, namely crude enzyme liquid, and preserving the crude enzyme liquid at 4 ℃.
Example 3 production method of Yeast Cycloastragenol polypeptide Mixed solution 1
1) Taking 1L of the enzyme solution in the embodiment 2, adding 40 g of astragalus extract containing 5% of astragaloside IV, and stirring and reacting at the temperature of 70-80 ℃ for 1-3 hours; obtaining reaction liquid;
2) then adding 15-30 times of water into the reaction liquid for dilution, then cooling to 25-40 ℃, and then sequentially adding carnosine (Cas:305-84-0), palmitic acid pentapeptide (Cas:214047-00-4) and acetyl hexapeptide-8 (Cas: 616204-22-9); obtaining mixed solution of yeast cycloastragenol polypeptide; storing at-20 deg.C;
wherein, in the mixed solution of yeast cycloastragenol polypeptide, the content of cycloastragenol is 0.01 percent,
the content of carnosine was 100ppm,
the content of the palmitic acid pentapeptide is 50ppm,
the content of acetyl hexapeptide-8 was 50 ppm.
Example 4 production method of Yeast Cycloastragenol polypeptide Mixed solution 2
1) Taking 1L of the enzyme solution in the embodiment 2, adding 60g of astragalus extract containing 5% of astragaloside IV, and stirring and reacting at the temperature of 70-80 ℃ for 1-3 hours; obtaining reaction liquid;
2) then adding 15-30 times of water into the reaction liquid for dilution, then cooling to 25-40 ℃, and then sequentially adding carnosine (Cas:305-84-0), palmitic acid pentapeptide (Cas:214047-00-4) and acetyl hexapeptide-8 (Cas: 616204-22-9); obtaining mixed solution of yeast cycloastragenol polypeptide; storing at-20 deg.C; wherein, in the mixed solution of yeast cycloastragenol polypeptide, the content of cycloastragenol is 0.02 percent,
the content of carnosine was 50ppm,
the content of the palmitic acid pentapeptide is 200ppm,
the content of acetyl hexapeptide-8 was 200 ppm.
Example 5 production method of Yeast Cycloastragenol polypeptide Mixed solution 3
1) Taking 1L of the enzyme solution in the embodiment 2, adding 50 g of astragalus extract containing 6% of astragaloside IV, and stirring and reacting at the temperature of 70-80 ℃ for 1-3 hours; obtaining reaction liquid;
2) then adding 15-30 times of water into the reaction liquid for dilution, then cooling to 25-40 ℃, and then sequentially adding carnosine (Cas:305-84-0), palmitic acid pentapeptide (Cas:214047-00-4) and acetyl hexapeptide-8 (Cas: 616204-22-9); obtaining mixed solution of yeast cycloastragenol polypeptide; storing at-20 deg.C; wherein, in the mixed solution of yeast cycloastragenol polypeptide, the content of cycloastragenol is 0.01 percent,
the content of carnosine was 100ppm,
the content of the palmitic acid pentapeptide is 100ppm,
the content of acetyl hexapeptide-8 was 100 ppm.
Example 6 temperature stability test of Yeast Cycloastragenol polypeptide mixture 1
Adding antiseptic into 1L yeast cycloastragenol polypeptide mixed solution 1, preserving at 70 deg.C and 30 deg.C respectively, sampling once per week for 12 weeks, and detecting contents of cycloastragenol, carnosine, pentapeptide palmitate and acetyl hexapeptide-8 by LC/MS respectively. The volume of the sample is up to 1L before each sampling. Experiments show that (fig. 1): when the mixed solution is stored at 70 ℃ for 12 weeks, the content of the core component cycloastragenol in the mixed solution is almost unchanged, the content of carnosine, palmitic acid pentapeptide and acetyl hexapeptide-8 is obviously reduced, and the maximum reduction amplitude is close to 35 percent, which is caused by the fact that the peptide cannot tolerate high temperature. And when the four-component health-care food is stored at 30 ℃ for 12 weeks, the content of the four active ingredients in the mixed solution is changed within 4 percent, and the use is not influenced.
Example 7 improvement of unevenness of skin texture of Mixed solution 1 of Yeast Cycloastragenol polypeptide
Chinese Han nationality women of 34 years old, 46 years old and 58 years old are respectively selected and applied to the face twice a week for 12 weeks by using 5% yeast cycloastragenol polypeptide mixed solution 1. The experimental results were observed with an electron microscope at 20 times magnification (fig. 2): after 12 weeks of use by women of different ages, the unevenness of the skin texture was improved and the skin became smoother.
Example 8 reduction of epidermal fine lines in Yeast Cycloastragenol polypeptide mixture 1
Two ages of 45 and 48 years old with obvious wrinkles on eyes and Chinese middle-aged females (Uygur family) were selected for the experiment, one group used placebo and one group used 5% yeast cycloastragenol polypeptide mixture 1, and the mixture was applied to the face, mainly around the eye, twice a week for 12 consecutive weeks.
The results show (fig. 3): the fine lines of the canthus of the group 1 are obviously improved by using 5% yeast ring astragaloside polypeptide mixed liquor.
Example 9 improvement of epidermal cytochrome and elasticity of Mixed solution 1 of Yeast Cycloastragenol polypeptide
Selecting Chinese Han women 50-60 of twenties, applying placebo (10 persons) and mixed solution 1 of placebo and 5% yeast cycloastragenol polypeptide (10 persons) on face, mainly near eye, twice per week for 12 weeks. According to the self-rating of the experimenter, the score is-4 to +4 (positive score is improvement, and negative score is deterioration) aiming at the senile plaque (color spot), the dark eye and the skin elasticity.
The results show (fig. 4): the 5% yeast cycloastragenol polypeptide mixed liquor has a certain effect of improving pigmentation, but has very obvious effect of improving skin elasticity.
Example 10 repair of skin Barrier function of Yeast Cycloastragenol polypeptide Mixed solution 1
The trans-epidermal water loss rate (TEWL) is the rate of water loss (g/h/m) per unit time through the epidermis2). The higher the TEWL, the more water lost per unit time and the weaker the skin moisturizing ability. Twenty-aged chinese han women (same volunteers as in example seven) at 50-60 years old were selected, one group with placebo (10), one group with placebo + 5% yeast cycloastragenol polypeptide cocktail 1 (10), face-painted twice weekly for 12 consecutive weeks, and tested for TEWL values every 4 weeks.
The results show that: the TEWL value of the population using the placebo and 5% yeast cycloastragenol polypeptide mixed solution is greatly reduced compared with the TEWL value of the population using only the placebo, and the yeast cycloastragenol polypeptide mixed solution has a good function of repairing skin barriers.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. A method for producing yeast cycloastragenol polypeptide mixed liquor is characterized in that: the method comprises the following steps:
1) inoculating Kluyveromyces lactis mutant strain XT82 into YPD liquid culture medium for culture and activation; then transferring the cells into a YPD liquid culture medium for fermentation, and centrifugally collecting the upper cells; the Kluyveromyces lactis mutant strain XT82 is named as Kluyveromyces lactis XT82 and is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: m2019724;
2) washing the collected cells with deionized water for 2-3 times, dissolving in deionized water to prepare suspension cells, crushing the cells, centrifuging, and collecting supernatant to obtain crude enzyme solution;
3) weighing the astragalus extract and the crude enzyme solution according to the weight-volume ratio of 40-60 g/L; stirring for reaction to obtain reaction liquid;
4) then adding water into the reaction liquid for dilution, then cooling to 20-60 ℃, and then sequentially adding carnosine, palmitic acid pentapeptide and acetyl hexapeptide-8; obtaining mixed solution of yeast cycloastragenol polypeptide; wherein, in the mixed solution of yeast cycloastragenol polypeptide, the content of cycloastragenol is 0.01-0.02%, the content of carnosine is 50-200ppm, the content of palmitic acid pentapeptide is 50-200ppm, and the content of acetyl hexapeptide-8 is 50-200 ppm.
2. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 1, characterized in that: in the step 1), the YPD liquid culture medium is prepared by the following steps:
sterilizing 10 g of the fermented extract and 20 g of peptone in 900ml of deionized water at 121 ℃ for 20 min; after cooling to about 60 ℃, 50ml of filtered and sterilized 40% glucose solution is added and mixed evenly.
3. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 1, characterized in that: in the step 3), the weight-volume ratio of the astragalus extract to the crude enzyme solution is 40-60 g/L; wherein the content of astragaloside IV in the radix astragali extract is 4-6%.
4. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 1, characterized in that: in the step 4), the stirring reaction conditions are as follows: the temperature is 70-80 ℃ and the time is 1-3 h.
5. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 1, characterized in that: in the step 4), the volume ratio of the reaction liquid to water is 1: 5-100.
6. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 5, characterized in that: in the step 4), the volume ratio of the reaction liquid to water is 1: 10-50 parts of; the temperature of the reaction solution was reduced to 20-45 ℃.
7. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 6, wherein: in the step 4), the volume ratio of the reaction liquid to water is 1: 15-30 parts of; the temperature of the reaction solution was reduced to 25-40 ℃.
8. The method for producing mixed solution of yeast cycloastragenol polypeptide according to claim 1, characterized in that: in the mixed solution of the yeast cycloastragenol and the polypeptide, the content of the cycloastragenol is 0.01 percent; the content of carnosine is 100 ppm; the content of the palmitic acid pentapeptide is 50 ppm;
the content of acetyl hexapeptide-8 was 50 ppm.
9. An application of the yeast cycloastragenol polypeptide mixed liquor in claim 1 in preparing products for resisting skin aging.
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| CN114177104A (en) * | 2021-11-25 | 2022-03-15 | 湖州中科锦肽生物科技有限公司 | Anti-aging face cream |
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| CN114177104A (en) * | 2021-11-25 | 2022-03-15 | 湖州中科锦肽生物科技有限公司 | Anti-aging face cream |
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